Transcription factors play important roles during cell fate determination and expression of tissue-specific genes that are necessary for normal organ development. Our previous studies revealed that transcription factors encoded by the Msx homeobox gene family play an important role in the development of ectodermal organs. Genetic studies in mice indicate that loss-of-function of the first member of the Msx gene family, the Msx1, affects tooth formation. We reported that (i) the Msx1 mutant mice exhibit a failure of tooth development and arrest at an early stage of development known as the bud stage and (ii) that Msx1 controls the expression of several downstream genes that are co-expressed with Msx1 during the bud stage of tooth development. Our current preliminary studies indicate that (i) Msx1 interacts in vitro and in vivo with several transcription factors that are co-expressed with Msx1 during bud stage of tooth development (ii) that Msx1 selectively binds in vivo to a specific target gene promoter, (iii) that Msx1 is modified at the posttranslational level by sumoylation in vivo, by Sumo1, (iv) that Sumo1 is co-expressed with Msx1 in the bud stage dental mesenchyme and that (v) Msx1 sumoylation by Sumo1 modifies the ability of Msx1 to interact with the tooth-specific, protein-protein interaction network and its target gene selection. These observations serve as the basis for our proposed hypothesis, that the molecular function of Msx1 transcription factor during early tooth development depends on the combinatorial action of a context dependent, tooth-specific transcription factor network and posttranslational modifications. To address this hypothesis, this proposal has the following research objectives (a) To validate the Msx1-interacting protein partners in vitro and in vivo (b) To determine how the Msx1 target promoter is regulated by the combinatorial action of Msx1 and Msx1-interacting protein complex(s) in living cells (c) To investigate how sumoylation of Msx1 contributes to regulation of genes in the developing tooth, by analyzing whether the Msx1- dependent protein-protein interaction network and target gene selection by Msx1 is modulated by the sumoylated or non-sumoylated state of Msx1. The fundamental understanding of how Msx1 regulatory protein functions will provide valuable insight on the function of Msx1 under normal conditions, on the transcriptional mechanism regulating early tooth development, on the pathogenetic mechanisms of syndromic or non-syndromic types of anodontia, and may provide valuable information towards treatment of these diseases and tooth regeneration. This study will also provide a potential general mechanism for understanding how cell-specific network of transcription factors achieve target gene selection. PUBLIC HEALTH RELEVANCE: This project aims at understanding the molecular mechanism by which Msx1 functions, using the developing tooth, as a model system. To accomplish this purpose, techniques of contemporary molecular biology will be employed to test the proposed hypothesis that the molecular function of Msx1 transcription factor during early tooth development depends on the combinatorial action of a context dependent, tooth-specific transcription factor network and posttranslational modifications.